Composition for promoting chondrocyte differentiation or treating cartilage diseases, containing KLF10 expression inhibitor, and method for promoting cartilage differentiation by using same

a technology of klf10 and expression inhibitor, which is applied in the direction of cell culture active agents, genetically modified cells, skeletal/connective tissue cells, etc., can solve the problems of difficult regeneration of damaged cartilage into original tissue of hyaline cartilage, difficult to achieve hyaline cartilage regeneration, and poor cartilage hypertrophy, so as to achieve excellent effect of promoting differentiation into chondrocytes and inhibiting cartilage hypertrophy

Active Publication Date: 2017-01-19
DONGGUK UNIV IND ACADEMIC COOPERATION FOUND
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0034]When expressed in a bone marrow stem cell to induce differentiation into a chondrocyte, the Krueppel-like factor 10 (KLF10) expression inhibitor of the present invention exhibits an excellent efficacy of promoting differentiation into a chondrocyte and inhibiting cartilage hypertrophy.

Problems solved by technology

Currently, for advanced degenerative arthritis, the standardized treatment is to remove the affected cartilage and bone and replace them with an artificial joint consisting of a metal(s) and a polyethylene, but the durability of such an artificial joint becomes an issue when it is implanted into a relatively young patient in his / her 60s or younger.
The development of damaged cartilage is caused by osteoarthritis which brings a traumatic loss or gradual destruction of articular cartilage tissue, and, despite the high incidence thereof, the regeneration of damaged cartilage into original tissue thereof, i.e., hyaline cartilage, is difficult, and not much is known about the molecular mechanism of cartilage regeneration.
Among them, conservative treatment such as medication is limited to the restoration of a limited number of functions by alleviating symptoms, and autologous chondrocyte implantation used for treating a traumatic loss of articular cartilage causes damage in the donor site by harvesting a bone-cartilage piece therefrom and the amount collectable is limited.
In addition, bone marrow drilling performed for osteoarthritis with moderate progression results in the regeneration of fibrous cartilage instead of the original cartilage tissue, i.e., hyaline cartilage, thus generating poor clinical results.
Moreover, the durability of an artificial joint can also be an issue when the artificial joint is implanted into a young patient, although artificial joint replacement is currently the standardized treatment for advanced osteoarthritis.
However, still, there is a lack of clear knowledge of cartilage formation in terms of the factors, environment, and the like.
However, until now, any reaction mechanism of IHH protein generation inhibition by PTHrP has not been explained in terms of a precise molecular biological mechanism.

Method used

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  • Composition for promoting chondrocyte differentiation or treating cartilage diseases, containing KLF10 expression inhibitor, and method for promoting cartilage differentiation by using same
  • Composition for promoting chondrocyte differentiation or treating cartilage diseases, containing KLF10 expression inhibitor, and method for promoting cartilage differentiation by using same
  • Composition for promoting chondrocyte differentiation or treating cartilage diseases, containing KLF10 expression inhibitor, and method for promoting cartilage differentiation by using same

Examples

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example 1

[0065]Analysis of Change in miRNA Expression with or Without Addition of PTHrP when Differentiation of Human Bone Marrow Stem Cell Into Cartilage is Induced

[0066]A miRNA microarray analysis was carried out to observe a change in a miRNA expression level as a result of a treatment with PTHrP when inducing the differentiation of human bone marrow stem cell into cartilage. First, while carrying out a treatment with TGF-β3 for total of 4 weeks to induce the differentiation of human bone marrow stem cells into cartilage, the cells were divided into a group additionally treated with PTHrP for the last 1 or 2 weeks and a group not treated with PTHrP, and RNA was separated from a cell pellet in which cartilage differentiation had been induced, and then, cDNA labeled with a Cy3-fluorescent dye was synthesized, from the RNA, for the entire miRNA. Then, the cDNA was hybridized on an Agilent human miRNA microarray chip coated with about 15,000 different DNA base sequences complementary to human...

example 2

[0069]Analysis of Cartilage Differentiation and Hypertrophy Inhibition Efficacies of Using Recombinant Lentivirus Expressing miR-892b

[0070]In order to prepare a recombinant lentivirus expressing miR-892b, first, a 340-bp DNA segment (that comprises pre-mature miR-892b acquired from human bone marrow stem cell DNA through genomic PCR) and a pCDH lentivirus vector were cut by being treated respectively with NheI and EcoRI restriction enzymes and were connected to each other to prepare a pCDH-CMV-miR-892b-EF1-copGFP recombinant vector as shown in FIG. 2. Then, the recombinant vector and a lentiviral packaging system comprising 3 types of packaging vectors (pLP1, pLP2, pVSVG) in a 1:1.5:1 ratio for lentivirus production were simultaneously transfected, in a 1:3 ratio, into 293FT cells. About 48 hours later, the culture medium was recovered to measure the concentration (titer) of the produced virus and was used for an experiment during which human bone marrow stem cells were subjected to...

example 3

[0081]Screening and Confirmation of miR-892b Target Genes

[0082]Binding sites of a KLF10 or WNT6 gene for miR-892b within a 3′UTR are as shown in FIG. 5A, wherein the KLF10 or WNT6 gene is predicted as a target gene of miR-892b based on the results of the above Example 2. A gene sequence comprising the binding sites of the KLF10 or WNT6 gene for miR-892b within a 3′UTR and a mutated base sequence within a critical binding site was synthesized to clone a pMIR-luciferase reporter vector (see FIG. 5B; WT, wild type; MUT, mutated). Also, luciferase reporter vectors prepared as described above and a miR-892b mimic or negative miRNA prepared by GE Healthcare Dharmacon Inc. were used to transfect a Hela cell, and, 48 hours later, the degree of target gene expression inhibition by miR-892b was examined. As appears in FIG. 5C, the results showed that, in the case of cells (WT-3′UTR) transfected with the normal KLF10 3′UTR, the rate of luciferase expression in a group transfected with miR-892b...

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Abstract

The present invention relates to a Krueppel-like factor 10 (KLF10) gene expression inhibitor promoting cartilage differentiation, and more specifically, to: a composition for promoting chondrocyte differentiation or treating cartilage diseases, containing a KLF10 gene expression inhibitor promoting cartilage differentiation and inhibiting the hypertrophy and dedifferentiation of chondrocytes; a cell therapeutic agent containing the composition; a method for promoting cartilage differentiation in bone marrow stem cells, comprising a step of expressing the composition in bone marrow stem cells; and a method for screening a chondrocyte differentiation promoter or a chondrocyte therapeutic agent. The present invention first examined the generation inhibition mechanism of indian hedgehog (IHH) protein of which the molecular biological mechanism has not yet been clearly examined, and ascertained that chondrocyte differentiation is promoted and chondrocyte hypertrophy is inhibited when chondrocyte differentiation is induced by expressing the KLF10 expression inhibitor in bone marrow stem cells. Therefore, the present invention has an advantage of enabling the use of bone marrow stem cells, which express a KLF10 expression inhibitor, as a chondrocyte therapeutic agent.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a Krueppel-like factor 10 (KLF10) gene expression inhibitor promoting chondrocyte differentiation, and, more particularly, to a composition for treating a cartilage disease or promoting chondrocyte differentiation, wherein the composition comprises a KLF10 gene expression inhibitor for promoting chondrocyte differentiation and inhibiting chondrocyte hypertrophy and dedifferentiation.BACKGROUND ART[0002]There have been many attempts to ease the difficulty of healing damaged articular cartilage, since, once damaged, the articular cartilage cannot be regenerated into original tissue. Currently, for advanced degenerative arthritis, the standardized treatment is to remove the affected cartilage and bone and replace them with an artificial joint consisting of a metal(s) and a polyethylene, but the durability of such an artificial joint becomes an issue when it is implanted into a relatively young patient in his / her 60s or younge...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/113C12Q1/68C12N5/077
CPCC12N15/1136C12N5/0655C12Q1/6883C12N2320/30C12N2310/141C12N2310/531C12N2501/60C12Q2600/136C12Q2600/158C12Q2600/178C12N2506/1353C12N15/113C12N2310/14C12N2501/65C12N2510/00
Inventor IM, GUN-ILLEE, JONG-MIN
Owner DONGGUK UNIV IND ACADEMIC COOPERATION FOUND
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